Chronic myeloid leukemia (CML) is driven by the BCR-ABL1 oncoprotein with constitutively active protein-tyrosine kinase activity, perturbing multiple signaling pathways. Although therapies with tyrosine kinase inhibitors (TKIs) can effectively treat early phase CML, relapses and emergence of TKI resistance are problematic, due to BCR-ABL kinase domain mutations and TKI unresponsive quiescent leukemic stem cells (LSCs). These observations point towards a need for alternate treatment strategies to prevent the development of resistant LSCs. We previously demonstrated that Abelson helper integration site-1 (AHI-1) is a highly deregulated protein in CML LSCs and that its WD40-repeat domain physically interacts with BCR-ABL, enhancing leukemia-initiating activity. AHI-1 also contains an SH3 domain, which mediates TKI resistance in LSCs. This domain interacts with dynamin-2 (DNM2) and forms a complex with BCR-ABL, to enhance the phosphorylation and activity of DNM2. The AHI-1-BCR-ABL-DNM2 complex is shown to regulate leukemic properties in patient LSCs, including increased ROS production, endocytosis and autophagy. Interestingly, deletion of the Ahi-1 SH3 domain (Ahi-1 SH3Δ) results in a defect in Ahi-1 localization, with most being present in the nucleus. To test whether Ahi-1 SH3 domain activity directly affects cytoplasmic anchoring and localization, we have generated two Ahi-1 mutants, using site-directed mutagenesis: a mutation in the key tryptophan residue (W939A) involved in SH3 domain binding and in a non-conserved surface residue (M906A), as a negative control, based on the crystal structure of the AHI-1 SH3 domain. Interestingly, the cytoplasm-to-nucleus signal ratio of Ahi-1 W939A was significantly reduced compared to the negative control or wildtype Ahi-1, as assessed by immunofluorescence and confocal microscopy (70% reduction, p<0.0001), indicating that changes in localization of Ahi-1 SH3Δ may result in disruption of the complex and allow for new interactions with nuclear proteins. Investigating changes in the proteome may help uncover downstream effects of the AHI-1-BCR-ABL-DNM2 complex and its biological role in mediating TKI resistance. Advanced antibody microarray analysis was then used to investigate differences in the proteome and phosphorylation landscape of BCR-ABL+ cells co-transduced with wildtype Ahi-1 or Ahi-1 SH3Δ. This system quantifies the differences in expression and phosphorylation states of key signaling proteins simultaneously, using 878 antibodies in duplicate. Twenty leads were identified by the following criteria: a large signal difference of at least 1.5-fold change, high signal strength for high expression, and low error between duplicates. These leads were validated by Western blot analysis and several of them were confirmed. Particularly, phosphorylation of cyclin-dependent kinase 1 (CDK1), a key player in cell cycle control and mitochondrial dynamics, was greatly reduced in cells expressing wildtype Ahi-1 compared to Ahi-1 SH3Δ, indicating that AHI-1-mediated phosphorylation changes in CDK1 may contribute to regulation of mitochondrial functions. Indeed, BCR-ABL-transduced cells co-expressing wildtype Ahi-1 showed increased mitochondria potential in response to TKI treatment or serum starvation, in MitoTracker analysis (p<0.05). However, this was not observed in BCR-ABL-transduced cells co-expressing the Ahi-1 SH3Δ mutant. A similar trend was also observed in immunofluorescence confocal microscopy analysis of the mitochondrial importer receptor, TOM20. To further study the role of DNM2 in mediating mitochondrial dynamics associated with AHI-1 and BCR-ABL, CRISPR-Cas9 mediated DNM2 knockdown was performed in TKI-resistant cells, using two different DNM2-targeting guide RNAs; these resulted in significant reduction in DNM2 (78% & 75%) in Western blot analysis. The knockdown cells showed a reduction in viability (60% reduction) and increased sensitivity to TKI treatment compared to the control (90% vs. 30% reduction) after 48 hours and changes in mitochondrial activity were also observed in these cells. These results support a role for mitochondrial dynamics in the AHI-1-BCR-ABL-DNM2 complex-mediated TKI response and that targeting key biological processes regulated by the AHI-1-BCR-ABL-DNM2 complex and its pathways may lead to new therapeutic strategies to overcome TKI resistance in CML.
Disclosures
Pelech: Kinexus Bioinformatics Corporation: Equity Ownership.
Characterization of Human Immunoglobulin (Ig) Isotype and IgG Subclass Response to Bartonella henselae Infection
